Abstract: The goal of this SBIR research program is to develop a novel small molecule inhibitor of lytic Epstein-Barr Virus (EBV) infection. EBV is a ubiquitous gamma-herpesvirus that has been classified by the World Health Organization as a human carcinogen. Vironika, with its consortium partners the Wistar Institute and Fox Chase Chemical Diversity Center, Inc., will develop a highly specific and potent inhibitor of EBV lytic (re)activation that will provide an important therapeutic strategy to treat EBV-associated diseases. EBV infection is associated with multiple human malignancies, including Burkitt's lymphoma, nasopharyngeal carcinomas, Hodgkin's lymphoma, gastric carcinomas, and immunoblastic B-cell lymphoma's during immunosuppression. Currently, no EBV-specific therapies exist that target lytic (re)activation, and therefore it remains impossible to effectively treat or prevent EBV-associated disease. The lytic infection depends on a viral encoded protein, ZTA, which functions in the (re)activation of the lytic virus cycle. The binding domain of this protein has been characterized structurally and biochemically, and serves as an ideal molecule for targeted small molecule inhibition of EBV infection. We have screened over 420,000 compounds in our primary HTS screen. Preliminary hits were filtered by counterscreen, and cherry picked and validated in triplicate single concentration assay formats. Among the 42 hits that passed these criteria, three chemotypes were identified. In this Phase 1 proposal, we will further validate these hits and generate focused libraries to advance a lead compound using medicinal chemistry methods and extensive biochemical and biological analysis to validate mechanism of drug action. In Phase 2, we will develop our advanced leads into a pre-clinical lead candidate. The ultimate goal of this SBIR program is to develop a novel small molecule therapeutic agent to treat lytic EBV infection and its associated malignancies.